Oh et al. Oncogenesis (2019) 8:27 https://doi.org/10.1038/s41389-019-0137-z Oncogenesis ARTICLE Open Access TBX3 represses TBX2 under the control of the PRC2 complex in skeletal muscle and rhabdomyosarcoma Teak-Jung Oh1, Abhinav Adhikari 2, Trefa Mohamad3, Aiysha Althobaiti2 and Judith Davie2 Abstract TBX2 and TBX3 function as repressors and are frequently implicated in oncogenesis. We have shown that TBX2 represses p21, p14/19, and PTEN in rhabdomyosarcoma (RMS) and skeletal muscle but the function and regulation of TBX3 were unclear. We show that TBX3 directly represses TBX2 in RMS and skeletal muscle. TBX3 overexpression impairs cell growth and migration and we show that TBX3 is directly repressed by the polycomb repressive complex 2 (PRC2), which methylates histone H3 lysine 27 (H3K27me). We found that TBX3 promotes differentiation only in the presence of early growth response factor 1 (EGR1), which is differentially expressed in RMS and is also a target of the PRC2 complex. The potent regulation axis revealed in this work provides novel insight into the effects of the PRC2 complex in normal cells and RMS and further supports the therapeutic value of targeting of PRC2 in RMS. Introduction phenotypes and the presence of myogenic markers such 4 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; Rhabdomyosarcoma (RMS) is the most common soft as myogenic regulatory factors (MRFs) , yet these factors tissue pediatric sarcoma, which is thought to largely arise appear to be inactive in RMS5. from the skeletal muscle lineage1. The more common The T-box family of transcription factors are highly form of the disease is the embryonal subtype (ERMS), conserved and related throughout all metazoan lineages. characterized by loss of heterozygosity at the 11p15 locus, They share a common DNA-binding domain known as a region which harbors insulin-like growth factor 2. the T-box motif and participate in diverse types of orga- Alveolar RMS (ARMS) is the more aggressive form of nogenesis and developmental regulation6. The T-box RMS that is characterized by t(2;13)(q35;q14) or t(1;13) motif binds to the core sequence GGTGTGA known as (q36;q14) translocations. The translocations result in the T-element7. Distinct from most members of the T- chimeric transcripts that fuse the 5′ portion of the paired box family, TBX2 is known as a potent transcriptional box proteins 3 or 7 (PAX3 or PAX7), including an intact repressor that functions in both embryonic development, DNA-binding domain, to the transactivation domain of a and if deregulated, tumorigenesis8. The oncogenic forkhead transcription factor (FKHR), creating novel potential of TBX2 was first identified by its ability to − − PAX3-FKHR (t(2;13)(q35;q14)) or PAX7-FKHR (t(1;13) bypass cellular senescence in a Bmi / mouse embryo (q36;q14)) fusion proteins2,3. RMS is diagnosed by fibroblast model9. TBX2 has been shown to promote cell observation of unique skeletal muscle cell morphology proliferation and block cellular senescence by repressing CDKN1A (p21), CDKN2A (p14/19ARF)10, and PTEN11. Another member of the T-box family, TBX3, is also Correspondence: Judith Davie ([email protected]) 1School of Molecular and Cellular Biology, University of Illinois at Urbana- characterized as a transcriptional repressor and develop- Champaign, Urbana, IL 61801, USA mental regulator12. TBX2 and TBX3 are highly related 2 Department of Biochemistry and Molecular Biology and Simmons Cancer and thought to have arisen from a gene duplication Institute, Southern Illinois University School of Medicine, Carbondale, IL 62901, 13 USA event . In the case of deregulation, TBX3 also acquires Full list of author information is available at the end of the article. © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Oncogenesis Oh et al. Oncogenesis (2019) 8:27 Page 2 of 17 the potential to become an oncogene in melanoma cells14. (RH30 and RH28) with an expression plasmid for TBX311. TBX3 has been found to be overexpressed in melanoma15 As anticipated, we observed that TBX3 was upregulated and several sarcomas16. TBX3 has been shown to promote (Fig. 1a). Upon the upregulation of TBX3, we found that tumor metastasis and the migration of breast cancer TBX2 was downregulated (Fig. 1b) in RH30, RH28, and cells17. In MCF-12A breast epithelial cells and B16 mouse RD cells. The degree of TBX3 overexpression in RMS melanoma cells, TBX3 has been shown to directly repress cells corresponded to the degree of TBX2 repression in TBX2 under the control of TGF-β118. Our lab has shown each cell line tested (Fig. 1a, b). The repression of TBX2 that TBX3 is expressed throughout skeletal muscle dif- by TBX3 was confirmed at the protein level in RD, RH28, ferentiation, whereas the expression of TBX3 in RMS cell and RH30 cell lines (Fig. 1c). For the RD2 cell line, RNA lines is almost completely abrogated11. results were inconsistent but the protein analysis con- The polycomb repressive complexes 1 and 2 (PRC1/2) firmed that TBX3 repression of TBX2 could be observed are critical in the precise and accurate regulation of in these cells as well (Fig. 1d). development in many physiological systems including To understand if TBX2 and TBX3 reciprocally repres- skeletal muscle19. PRC1 and PRC2 work synergistically sed each other’s expression, we asked if TBX2 could also and indispensably together for transcriptional repression repress TBX3. RMS cell lines were transiently transfected of target gene by ubiquitylation of histone H2A lysine 119 with a shRNA construct against TBX227 and assayed for residue and methylation of histone 3 lysine 27 (H3K27) expression of TBX2 and TBX3. As anticipated, we residues, respectively20. The catalytic subunit of PRC2 is observed that TBX2 was depleted (Fig. 1e), but saw no enhancer of zeste 2 (Ezh2)21. upregulation of TBX3 (Fig. 1f). Thus, depletion of TBX2 Many studies have revealed that EZH2 is overexpressed does not upregulate TBX3. In fact, TBX3 was even more in a large number of cancers22. In RMS, pharmacological strongly downregulated in the absence of TBX2, indicat- inhibition of EZH2 in ERMS resulted in reduced pro- ing that TBX2 may play a role in activating TBX3 in RMS. liferation and pharmacological inhibition or depletion of Our data showed that TBX3 represses TBX2 in RMS cell EZH2-induced myogenic differentiation in these cells23. lines so we asked if this expression correlation could also EZH2 depletion in ARMS has also been shown to inhibit be observed in additional sarcomas using available patient proliferation and initiate apoptosis24. JARID2, a founding data. Sarcoma expression data for TBX3 and TBX2 were member of Jumonji family of proteins, is known to be a downloaded from The Cancer Genome Atlas (TCGA) substoichiometric component of PRC2 that appears to (https://cancergenome.nih.gov/) database using GDAC function in targeting PRC2 activity25. JARID2 is highly Firehose. Two cohorts of the top one-third TBX3 and expressed in ARMS and contributes to the inhibition of TBX2 expressing samples were combined and duplicates differentiation in ARMS cells26. samples were removed. A correlation analysis was per- In this work, we show that TBX3 represses TBX2 under formed and a scatter plot was generated for TBX3 and the control of the PRC2 complex in RMS and skeletal TBX2 expression (Fig. 1g). TBX2 and TBX3 were found to muscle. RMS cells and proliferating skeletal muscle cells be inversely correlated (Spearman correlation coefficient contain high levels of EZH2 that represses TBX3 and (rs) = −0.5173, p < 0.0001, N = 113), which was also evi- allows TBX2 expression. Depletion of EZH2 upregulates dent from two distinct clusters on the heatmap with TBX3 which then down regulates TBX2. We also show opposite expression (N = 263) (Fig. 1h). that the early growth response gene EGR1 is correlated To characterize the cellular effects of TBX3 in RMS, we with the induction of differentiation and repressed by generated stable RH30 cell lines expressing TBX3 and PRC2 in ARMS. Discovery of this novel PRC2-TBX3- three independent clonal isolates were used for further TBX2 genetic axis has important implications for under- analysis. Expression of exogenous TBX3 was confirmed at standing the mechanisms that drive proliferation and both the mRNA level (Fig. 2a) and the protein level (Fig. differentiation in RMS and skeletal muscle. 2c). We found that stable overexpression of TBX3 resul- ted in high levels of TBX3 mRNA expression (Fig. 2a) and Results repressed TBX2 mRNA expression (Fig. 2b). The repres- TBX3 represses TBX2 sion of TBX2 by TBX3 could also be observed at the We have previously shown that TBX2 is highly protein level (Fig. 2c). TBX3 was detected with antibodies expressed in RMS while TBX3 is not11,27. In skeletal against TBX3 and the V5 epitope tag only present on muscle, TBX2 is expressed in proliferating myoblasts, but exogenous TBX3. In each case, the degree of over- sharply downregulated upon differentiation while TBX3 is expressed TBX3 correlated to the degree of TBX2 expressed throughout myogenesis and highly expressed repression, confirming that TBX3 represses TBX2.